Plasma Oxytocin Variation in Families with Children with Autism Spectrum Disorders

Background:

Studies have reported genetic linkage and association with autism for the oxytocin receptor gene (OXTR) within independent samples. Our laboratory found overall association with autism and the rs2254298 single nucleotide polymorphism in OXTR (Jacob et al 2007).   Children with autism were reported to have lower average levels of blood oxytocin (OT) in comparison to typically developing children matched for age (Modahl et al 1998).  OT blood levels may serve as an intermediate phenotype that will help identify autism related genes.

Objectives:

To utilize enzyme immunoassay (EIA) methods to measure oxytocin in the blood of children with autism spectrum disorder (ASD) and their family members. Examining neurochemical heterogeneity in children with ASDs, will allow us to identify children with or without disruptions in OT and to explore relationships to genetic variation.  For clinical phenotype, we focused on non-verbal social communication given previous results in our lab and others (Brune et al 2007, Green et al 2001, Modahl et al 1998).

Methods:

We are investigating a growing sample (age 3 to 50 years) who meet clinical, Autism Diagnostic Interview-Revised (ADI-R), and Autism Diagnostic Observation Schedule (ADOS) criteria for ASD and their families.  Children completed extensive diagnostic testing including IQ measures and physical exams.  Blood was collected in chilled glass tubes containing disodium EDTA and kept on crushed ice.  The samples were centrifuged at 3500 rpm for 10 min in a freezing centrifuge 4 degree C. Plasma samples were divided into storage vials and stored at  -80 degree C.  OT was measured using the enzyme immunoassay (EIA) kit developed by Assay Designs, Inc. (Ann Arbor, MI). Assay Designs reported cross-reactivity for similar neuropeptides found in mammalian sera at less than 0.001% and a minimum detection limit of 11.7 pg/ml. EIA samples were diluted 8 fold and manufacturer’s instructions were followed (Carter et al. 2007).  OT values greater than 2 SD from the mean were excluded from descriptive statistics and assays will be re-run.  Given OT distribution, we used nonparametric statistics.

Results:

Preliminary results were obtained for 28 probands and their families with EIA. Mean OT from probands was 442 pg/ml (SD=555), from mothers was 409 pg/ml (SD=272), from fathers was 314 pg/ml (SD=99), and from 14 siblings was 204 pg/ml (SD=141).  Further analyses and quantification will be required to examine relationships between families and probands versus non-affected siblings.  Preliminary results show a positive trend between proband OT levels and ADI-R “failure to use nonverbal communication to regulate social interaction” sub-domain scores (Kendall tau, p=.06).  This was the single subdomain examined.  Future analyses will examine patterns of OT levels within families and the relationships between phenotype and genotype.

Conclusions:

Preliminary results show that plasma oxytocin measured by EIA show a broad range of values and a skewed distribution.  Our goal is to establish a methodology that will facilitate studying OT as an intermediate phenotype within ASD.  This may be correlated with observable behavioral differences in phenotype.  It will also allow us to connect phenotypic differences to genetic associations with autism and oxytocin-related genes.